&nbsp;红细胞膜相关蛋白通过IL-6/STAT3/ROR-γt 信号通路抑制小鼠实验性自身免疫性脑脊髓炎中Th17细胞分化

何可可; 李远迪; 文廷浩; 朱婕; 高杰; 胡蓉; 韩锋; 苏敏

doi:10.16098/j.issn.0529-1356.2023.05.006

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解剖学报 ›› 2023, Vol. 54 ›› Issue (5) : 538-545. DOI: 10.16098/j.issn.0529-1356.2023.05.006

细胞和分子生物学

红细胞膜相关蛋白通过IL-6/STAT3/ROR-γt 信号通路抑制小鼠实验性自身免疫性脑脊髓炎中Th17细胞分化

何可可¹ 李远迪¹文廷浩¹ 朱婕¹ 高杰^1,3 胡蓉^1,3韩锋⁴ 苏敏^1,2*

作者信息 +

Erythrocyte membrane-associated protein inhibiting Th17 cell differentiation via IL-6/STAT3/ROR-γt signaling pathway in experimental autoimmune encephalomyelitis mice encephalomyelitis mice

HE Ke-ke¹ LI Yuan-di¹ WEN Ting-hao¹ ZHU Jie1 GAO Jie^1,3HU Rong^1,3 HAN Feng⁴ SU Min^1,2*

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摘要

目的探讨小鼠实验性自身免疫性脑脊髓炎（EAE）模型中，外源性和内源性红细胞膜相关蛋白（ERMAP）通过白细胞介素6/信号转录及转录激活因子3（IL-6/STAT3）/维甲酸相关孤核受体γt/（ROR-γt）信号通路对辅助性T细胞17（Th17）细胞分化的影响。方法流式细胞术验证不同浓度ERMAP-Ig融合蛋白功能；琼脂糖凝胶电泳鉴定ERMAP基因敲除小鼠。流式细胞术检测ERMAP-Ig融合蛋白在体外对Th17细胞分化影响。40只6周龄普通C57BL/6小鼠随机分为2组建立EAE模型：对照（control-Ig和ERMAP-Ig组，每组20只；记录临床评分；流式细胞术检测EAE小鼠体内Th17细胞分化情况。40只6周龄已鉴定的野生型和ERMAP基因敲除小鼠分为2组建立EAE模型：ERMAP+/+和ERMAP-/-组，每组20只。记录临床评分；脊髓HE和固蓝（LFB）染色并进行组织学半定量评分。流式细胞术检测IL-17A+CD4+ T细胞百分比；Western blotting检测IL-6、白细胞介素17（IL-17）、STAT3、磷酸化STAT3（p-STAT3）、ROR-γt蛋白水平表达；Real-time PCR检测IL-17、肿瘤坏死因子α（TNF-α）、IL-6、STAT3和ROR-γt的mRNA表达；ELISA检测细胞水平IL-17和TNF-α的表达。结果 1.外源性ERMAP-Ig融合蛋白抑制体内外Th17细胞分化且减轻小鼠EAE症状。2.与对照组相比，ERMAP-/-组EAE小鼠炎性浸润和脱髓鞘症状加重，Th17分泌IL-17A增加。3.内源性ERMAP基因敲除后，IL-17、TNF-α、IL-6、STAT3及ROR-γt表达均增加。差异均有统计学意义（P<0.05）。

结论 ERMAP可能通过IL-6/STAT3/ROR-γt信号通路调控Th17细胞分化，参与小鼠EAE发生发展。

Abstract

Objective To explore the effect of exogenous and endogenous erythrocyte membrane-associated protein (ERMAP) on helper T cell 17(Th17) cell differentiation through interleukin 6/signal transducers and activators of transcription 3/retionoid-related orphan nuclear receptor-γt(IL-6/STAT3/ROR-γt) signal pathway in the mouse model of experimental autoimmune encephalomyelitis (EAE). Methods Using flow cytometry to verify the function of ERMAP-Ig fusion protein at different concentrations; Agarose gel electrophoresis was performed to identify ERMAP knockout mice. Flow cytometry was performed to detect the effect of ERMAP-Ig fusion protein on Th17 cell differentiation in vitro. Forty 6-week-old normal C57BL/6 mice were randomly divided into 2 groups to establish EAE models, control-Ig and ERMAP-Ig groups, with 20 mice in each group; Clinical scores were recorded; Flow cytometry was performed to detect Th17 cell differentiation in EAE mice in vivo. Forty 6-week-old identified wild-type and ERMAP knockout mice were divided into 2 groups to establish EAE models. Identified wild-type and ERMAP knockout mice were divided into 2 groups to establish EAE models, ERMAP+/+ and ERMAP-/- groups, with 20 mice in each group. Clinical scores were recorded; Spinal cord HE and luxol fast blue(LFB) staining and histological semi-quantitative scoring were performed. Flow cytometry was performed to detect the percentage of IL-17A+CD4+ T cells; Western blotting was performed to detect the expression of IL-6, IL-17, STAT3, p-STAT3, and ROR-γt protein levels; Real-time PCR was performed to detect the mRNA expression of IL-17, TNF-α, IL-6, STAT3, and ROR-γt; ELISA was performed to detect the expression of IL-17 and TNF-α at the cellular level was detected by ELISA. Results 1. Exogenous ERMAP-Ig fusion protein inhibited Th17 cell differentiation and attenuated EAE symptoms in mice. 2. Compared with the control group, ERMAP-/- EAE mice showed increased inflammation and demyelination symptoms and increased Th17 secretion of IL-17A. 3. Endogenous ERMAP knockdown resulted in increased expression of IL-17, TNF-α, IL-6 STAT3, and ROR-γt. All differences were statistically significant(P<0.05). Conclusion ERMAP may regulate Th17 cell differentiation through the IL-6/STAT3/ROR-γt signaling pathway and is involved in the development of EAE in mice.

导出引用

何可可李远迪文廷浩朱婕高杰胡蓉韩锋苏敏. 红细胞膜相关蛋白通过IL-6/STAT3/ROR-γt 信号通路抑制小鼠实验性自身免疫性脑脊髓炎中Th17细胞分化[J]. 解剖学报. 2023, 54(5): 538-545 https://doi.org/10.16098/j.issn.0529-1356.2023.05.006

HE Ke-ke LI Yuan-di WEN Ting-hao ZHU Jie GAO Jie HU Rong HAN Feng SU Min.

Erythrocyte membrane-associated protein inhibiting Th17 cell differentiation via IL-6/STAT3/ROR-γt signaling pathway in experimental autoimmune encephalomyelitis mice encephalomyelitis mice

[J]. Acta Anatomica Sinica. 2023, 54(5): 538-545 https://doi.org/10.16098/j.issn.0529-1356.2023.05.006

中图分类号： R34

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